Fabrication of Biocompatible, Functional, and Transparent Hybrid

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Fabrication of Biocompatible, Functional, and Transparent Hybrid Films Based on Silk Fibroin and Epoxy-silane for Biophotonics Robson Rosa da Silva, Mauricio Cavicchioli, Lais Rocalho de Lima, Caio Gomide Otoni, Hernane da Silva Barud, Silvia Helena Santagneli, Agnieszka Tercjak, André Capaldo Amaral, Renata Aquino Carvalho, and Sidney José Lima Ribeiro ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06061 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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ACS Applied Materials & Interfaces

Fabrication of Biocompatible, Functional, and Transparent Hybrid Films Based on Silk Fibroin and Epoxy-Silane for Biophotonics Robson R. da Silva,1* Maurício Cavicchioli,1 Laís R. Lima,1 Caio G Otoni,2 Hernane S. Barud,3 Silvia H. Santagneli,1 Agnieszka Tercjak,4 André C. Amaral,3 Renata A. Carvalho,3 Sidney J. L. Ribeiro1 1

Institute of Chemistry, São Paulo State University, Araraquara , São Paulo 14801-970, Brazil 2

National Nanotechnology Laboratory for Agribusiness, EMBRAPA-CNPDIA, São Carlos, São Paulo 13560-970, Brazil

3

Centro Universitário de Araraquara – UNIARA, Araraquara, São Paulo 14801-320, Brazil 4

Group `Materials + Technologies´ (GMT), Department of Chemical and Environmental

Engineering, Engineering College of Gipuzkoa, University of the Basque Country (UPV/EHU), Plaza Europa 1, 20018 Donostia-San Sebastián, Spain e-mail: [email protected] KEYWORDS: Photonics, hybrids, silk fibroin, GLYMO, Glycidyl 3-(trimethoxysilyl)propyl ether

Abstract In this work we explored the fabrication of flexible and transparent hybrids of silk fibroin and epoxy modified siloxane for photonic applications. It is well known that regenerated silk fibroin (SF) solutions can form free-standing films with high transparency. Although SF has a restricted number of chemically reactive side groups, the main issues of as-casted pristine SF films regard the high solubility into aqueous media, brittleness, and low thermal stability. The design of SF films with enhanced functionality but yet highly transparent triggers new opportunities on ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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broader range of applications in biophotonics. Here we present a simple, functional, yet remarkably versatile hybrid material derived from silica sol-gel process based on SF protein and (3-glycidyloxypropyl)trimethoxysilane (GPTMS), an organically modified silicon-alkoxide owning a reactive terminal epoxy group. Specifically, we investigated the effect of the addition of GPTMS into silk fibroin solutions on the processability, morphology, crystallinity, mechanical and optical properties of the resulting hybrid films. Highly transparent (ca. 90 %) and flexible free-standing hybrid films were achieved. Cell viability assays revealed that the hybrid films are non-cytotoxic to rat osteoblast cells even at high GPTMS content (up to 70 wt%). The hybrid films showed enhanced thermal stability and were rich in organic (epoxy) and inorganic (silanol) functional groups according to the content of GPTMS. We also evaluated the successful preparation of

highly-quality optical red emissive SF hybrid films by loading YVO4:Eu3+

nanoparticles at low concentration (< 5 wt%). A meaningful description of the hybrid film structure is reported from the combination of scanning electron and atom-force microscopies, vibrational spectroscopy, solid state NMR, and X-ray diffraction analyses. 1. Introduction A great deal of the ongoing efforts has been addressed towards the fabrication of optical materials derived from natural polymers.1 In the last decade, silk fibroin (SF) – a protein found in spider cobweb fibers and silkworm cocoons, among others – emerged as a suitable natural polymer for optics. In particular, SF films display high transparency (ca. 95%) across the visible range,2,3 low surface roughness,4 biocompatibility, and tunable biodegradability.3,5 Those features make SF films interesting nature-based materials for biophotonics.6,7 SF is primarily composed by amino acids with poorly chemically reactive residues. To date, Glycine (Gly), Alanine (Ala) and Serine (Ser) account to more than 80 mol% of the total composition. Tyrosine (Tyr) and Tryptophan (Tryp) aromatic residues account to less than 5 mol%.8 Transparent SF films can be easily prepared by casting SF aqueous solution over smooth or patterned surfaces.9,10 For instance, SF aqueous solution can be obtained by dissolving degummed native SF fibers in concentrated chaotropic salt solution such as LiBr, CaCl2/ethanol/water,11 LiSCN12 or ionic liquids.13 In general, pristine SF films are brittle and water-soluble and present poor thermal stability. In addressing these issues, insoluble SF films with improved mechanical and thermal properties have been achieved by subjecting pristine SF ACS Paragon Plus Environment

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films to post-treatments, including i) physical stretching;14 ii) water vapor annealing;15 iii) immersion in polar organic solvents;16 and iv) ultraviolet irradiation.12 In addition, the incorporation of organic and inorganic components such as plasticized blends,17 layered clays,18 reinforcing fibers,19 cross-linkers,20 and nanoparticles7,21 have been evaluated on the development of strategies to achieve transparent SF hybrid films with ameliorated chemophysical properties. Beyond that, several strategies have been devoted to introduce functional groups to SF seeking to develop new pathways, in which specific (bio)molecules can be further immobilized (e.g. antibodies, enzymes, etc.). Such strategies range since simple doping to sophisticated multi-step chemical modifications of aromatic residues. For example, many attempts have been made to modify Tyr residues such as enzyme-catalyzed with tyrosinase,22 cyanuric chloride-activated coupling,23 or sulfonation with chlorosulfonic acids.24 For instance, Kaplan’s group has successfully modified Tyr residues with sulfonate moiety with diazonium coupling reaction enabling the further functionalization with aniline derivative shaving amine, ketone, carboxylic acid or alkyl functional groups.25 Nevertheless, those strategies only embrace limited functionalization in light of the restricted amount of Tyr residues present in SF backbone. The sol-gel route is a versatile strategy to prepare hybrid materials with wealthy of functional groups. Broadly speaking, the sol-gel method combines inorganic and organic components under low temperature by hydrolytic polymerization of metal or nonmetal alkoxide precursor M(OR)n (M= Si, Ti, Zr, etc.). Presently, a myriad of sol-gel precursors displaying reactive functional groups are available. Recently, the sol-gel route has been used to fabricate transparent, mechanically robust SF hybrid films by the incorporation of (3-aminopropyl)trimethoxysilane, a silane alkoxide precursor containing an amine functional group.26 In an effort to extend the fabrication of functional SF hybrids, we investigated the preparation of flexible, transparent free-standing hybrids of SF and (3-glycidyloxypropyl)trimethoxysilane (GPTMS). GTPMS is an organically modified alkoxide silane, which has a terminal epoxy group. Besides the homogeneous incorporation of inorganic silica network into the SF polymeric chain, epoxy groups lead to polyethylene oxide (PEO) chains via photo, thermal and upon alkaline/acid catalytic polymerization. In addition, the GPTMS epoxy-ring can be used as coupling agent to covalently bind organic and inorganic networks. Due to such peculiarities, hybrids derived of GPTMS show exceptional optical properties for the fabrication of photonic devices such as planar waveguides,27,28 optical limiters,29 and nonlinear optical materials.30

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In the present contribution, we have studied the preparation of highly transparent (ca. 90 %) and flexible free-standing films comprising high GPTMS contents (up to 70 wt%). Benefiting from this, we also investigated the fabrication of functional luminescent lanthanide films by doping transparent free-standing SF/GPTMS hybrids with red-emitting Eu3+ based nanoparticles on the pursuit of evaluate their potential use in optical applications. It has been extensively demonstrated that the sol-gel process is a feasible approach to tailor hybrid films with trapped Eu3+ fluorescent nanoparticles in order to produce photonic materials like fiber and planar devices. The well-known advantages of using narrow line-like emission from long-lived Eu3+ excited states have been recently exploited by some of us to design an optical sensor of hepatits C.31 The detection was performed by evaluating the molecular recognition of antibody-antigenic peptide through changes in the luminescent properties of YVO4:Eu3+ nanoparticles incorporated on layer-by-layer SF films. Noteworthy, luminescent lanthanide nanoparticles are kinetically stable to acid/base and either solvolytic conditions and allow us to design optically active, functional films that fulfill the requirements of transparency and stability that are highly desired in photonic applications. To the best of our knowledge, there have been no studies reported on the multifunctional silk fibroin modified with epoxy groups fashioned on transparent freestanding films. There are several advantages over the use of epoxy-rich and luminescent probes in SF protein based transparent solid substrates. Transparent epoxide-activated solid supports enable i) easy and multipoint covalent immobilization of biological macromolecules (e.g. enzymes, proteins, antibodies) by reaction with nucleophiles such amines, hydroxyl and sulfhydryls groups;32 ii) close contact between the support surface and target (e.g. biomolecules, chelates) due to short length of the epoxy silane linker bearing, for example, minimal conformational changes of immobilized biological macromolecules and improvement of stability/activity/selectivity;33 iii) potential use in regenerative medicine to monitor cell growth using light. Light is an important tool to translate data for sensing, instruction, and visualization of biological tissues and light sensitive biomolecules;34 iv) feasible storage and transport of luminescent probes and biological macromolecules in solid state allowing long shelf-life and having potential application in realtime monitoring of analyte-induced luminescence switching in different optical sensor constructs.35


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2. Experimental 2.1.Materials Silk cocoons produced by Bombyx mori silkworms were supplied by Bratac, Fiação de Seda S.A.

(Bastos,

Brazil).

Na2CO3,

Na2O,

NaVO3,

CaCl2·2H2O,

Ethanol

P.A,

(3-

glycidyloxypropyl)trimethoxysilane, and tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Y2O3 99% and Eu2O3 99% were purchased from Lumintech. Bovine serum, Penicillin, and Streptomycin were purchased from Vitrocell.

2.2.Preparation of aqueous SF solution SF was extracted from silk cocoons. The method was based on a previous report.8 Five grams of silk cocoon pieces were degummed with 2 L of 0.02 M Na2CO3 solution at 100 °C for 30 min. The SF fibers were washed thoroughly with distilled water and allowed to dry at 40 °C. Afterwards, dried SF fibers were dissolved in ternary system solvent, CaCl2:H2O:ethanol with 1:8:2 molar ratio pre-heated at 80 °C for 1 h. SF fiber concentration was 250 g L−1. The resulting viscous solution was dialyzed against Milli-Q ultrapure water in a proportion of 1:80 (v/v) for 6 times throughout 48 h. The purification of dialyzed SF aqueous solution was proceeded by centrifugation at 20,000 rpm for 20 min to remove non-dissolved impurities. An aqueous SF solution with final concentration of 3.7 wt% was achieved and stored at 4 °C before further use.

2.3.Preparation of free-standing silk fibroin/(3-glycidoxypropryl)trimethoxysilane hybrid films SF films were obtained by casting 3 mL of pristine 3.7 wt% SF solution in polystyrene dishes (∅= 30 mm) and allowing to dry at room temperature for 48 h. The SF/GPTMS (SFGPTMS) hybrid films were fabricated by introducing 3 mL of 3.7 wt% aqueous SF solution followed by the addition of pure GPTMS. The mixture was slightly stirred with the help of a glass rod during 5 min and transferred to polystyrene dishes (∅= 30 mm). The films were allowed to dry at 40 ºC during 48 h. Films with GPTMS content lower than 70 wt% were free-standing. The samples



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were named as SFGPTMS x% with x= 10-70% indicating different GPTMS mass percentage contents. SFGPTMS 0% corresponds to the blank or pristine SF sample (unmodified). 2.4.Preparation of YVO4:Eu3+ nanoparticles Luminescent YVO4:Eu3+ nanoparticles were prepared as previously reportde.31 Sodium orthovanadate (Na3VO4) was prepared by reacting sodium oxide (Na2O) with sodium metavanadate (NaVO3) at 700 °C under a heating rate of 14 °C min−1 in a nitrogen atmosphere.36 The mixture was further kept at this temperature for 4 h and then cooled to 200 °C. After 10 h, the mixture was allowed to cool down to room temperature and the resulting white powder (Na3VO4) was dissolved in water. Aqueous solutions of Y(NO3)3 1 mol L−1 (9 mL) and Eu (NO3)3 1 mol L−1 (1 mL) were mixed with 5 mL of a sodium citrate solution 2 mol L−1 under vigorous stirring at room temperature. A white milky suspension was immediately formed upon lanthanide citrate precipitation. Afterwards, 50 mL of an aqueous solution of Na3VO4 0.2 mol L−1 was added. The mixture became transparent and was stirred for 45 min at 60 ºC leading to the formation of citrate ionstabilized YVO4:Eu3+ nanoparticles. Subsequently, the solution was cooled and dialyzed against Milli-Q ultrapure water for 72 h.37

2.5.Preparation of luminescent silk fibroin/(3-glycidyloxypropyl)trimethoxysilane hybrid films Luminescent SFGPTMS hybrid film was fabricated by mixing 3 mL of aqueous SF solution 3.7 wt% with 26 µL of GPTMS during 5 min (ca. SFGTPMS 20%). Afterwards, an aqueous suspension of YVO4:Eu3+ nanoparticles was added to the mixture under slight stirring. The mixture was then transferred to polystyrene dishes (∅= 30 mm). The content of YVO4:Eu3+ nanoparticles comprised 5 wt% of the overall composition.31 The mixture was dried at 40 ºC for 48 h in an oven.

2.6.Cytotoxicity assay The SF hybrid films needed to be prepared prior to the cytotoxicity assay. Pristine SF and SFGPTMS hybrid films were weighed, minced, immersed in cell culture Dulbeccos Modified Eagle's Medium (DMEM-Vitrocell) supplemented with 10% fetal bovine serum (FBS) and


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antibiotics (Penicillin and Streptomycin), and subjected to intense mechanical agitation for 24 h. After this step, all samples were removed and the resultant medium was filtered (bacteriological filter 22 µm-CASVI) and placed in cell culture plate wells (96 wells) containing osteoblasts derived from calvaria of neonatal rats (cell lineage OSTEO-1) in density 15.10³ cells well−1. Osteoblast cells are routinely used in research to study the cytotoxicity of SF derivative materials. The plates were kept in cell culture incubator (Panasonic-CO² incubator MOC-19 AIC-UV) at 37 °C, humidified atmosphere containing 5% CO2 and 95% air for 48 h. Cell viability was then determined by a colorimetric method MTT (tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide). Subsequently, culture medium was removed from the wells, which were washed with phosphate buffered saline (1X PBS). Aliquots of 100 µL of MTT were added to each well and the cells were kept in culture conditions for 4 h. After this period, they were added by 200 µL of isopropyl alcohol and the well content was mechanically homogenized until complete formazan solubilization. The absorbance optical density values (OD) were obtained using a spectrophotometer (Polaris-CELER) at 570 nm. The resulting measurement relative to SF and SFGPTMS membranes, performed in triplicates for each group, were converted into percentages of cell viability in relation to control group and subjected to analysis of variance (ANOVA-ONE WAY) at 5% level of significance. The control group consisted of cultured cells in standard medium that was not previously exposed to the films. Values of the pristine SF or SFGPTMS hybrid samples significantly lower than control were assigned to cytotoxicity. 2.7.Instrumentation Film transmittance spectra of films were acquired on a Pelkin Elmer LAMBDA 1050 UV/Vis spectrophotometer at wavelengths in the range of 200 to 800 nm. Film thicknesses (w) and refractive indexes (n) of SFGPTMS films were measured on a Metricon prism coupler model 2010/M using a prism with n range of 1.20 to 2.02 and a 543-nm laser as light source by m-lines technique. Prism coupling technique provides excellent experimental accuracy (instrumental routine index accuracy of ±0.0005) due to the increased light-material interaction inherent to guided mode. At least 5 specimens of each composition were analyzed. Scanning electron microscopy (SEM) was used to analyze the surface morphology of films. The images were obtained on a field emission JEOL equipment model 7500F working at



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accelerating voltages below 5 KV. Films were placed directly on carbon conductive tape and then carbon-coated prior to SEM analysis. The morphology of YVO4:Eu3+ nanoparticles was investigated by transmission electron microscopy (TEM) technique. TEM images were acquired on a JEOL JEM 3010 high-resolution transmission electron microscope operating at 300 kV. A diluted suspension of YVO4:Eu3+ nanoparticles was deposited over carbon-coated copper grids and allowed to dry at room temperature. Samples were analyzed without any further treatment. The surface topography and roughness of films were evaluated by atomic force microscopy (AFM) on an Agilent 5500 microscope operating in intermittent contact mode under ambient conditions as well as using conventional silicon multipurpose cantilevers. All images were rendered using Gwyddion AFM analysis software. Fourier transform infrared spectroscopy (FTIR) was carried out on a Spectrum 2000 FTIR Perkin Elmer spectrophotometer, equipped with a multiple reflection, horizontal MIRacle attenuated total reflectance (ATR) attachment (ZnSe crystal). The spectra were collected at wavenumbers ranging from 4000 to 700 cm−1 with an accumulation of 128 scans and a resolution of 4 cm−1. Raman spectra were recorded using a Raman Horiba Jobin Yvon 118 model LabRAM HR 800 spectrometer, operating with He–Ne 632.81 nm laser through a 100x objective lens and dispersed by a diffraction grating (600 lines mm−1) onto a cooled charge-coupled device (CCD) detector. FTIR and Raman measurements were used to investigate the functionalization and conformation changes of SF films. The solid state 13C-1H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra were recorded using a Varian INOVA 500 spectrometer equipped with a 4-mm, T3-MAS probe. The experiment was carried out at a spinning rate of 5 kHz, using an excitation pulse length of 3.2 µs under continuous-wave (CW) 1H decoupling, and a 5-s delay between repetitions. Chemical shifts were reported in relation to TMS referencing standard. MAS-NMR measurements were used to elucidate the hydrolysis extension of epoxy precursor and conformation of SF in hybrid films. X-ray diffractograms were registered on a Rigaku Rint 2000 diffractometer, under the following experimental conditions: 40 kV, 120 mA, 4 ° ≤ 2θ ≤ 70 °, step scan of 0.005 ° 2θ, λCukα monochromatized by a graphite crystal, divergence slit= 1/2 mm, and reception slit= 0.3 mm.


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X-ray diffraction (XRD) was used to elucidate the crystal structure of fibroin in pristine SF and hybrid films. For the measurement of mechanical properties, films were shaped into ca. 50-mm-long, 5-mmwide strips and allowed to equilibrate at 50% RH in a desiccator containing a saturated Mg(NO3)2 solution for 48 h prior to thickness measurements with a digital micrometer (Mitutoyo Manufacturing, Japan). Then, films were submitted to tensile assay on a DMA Q800 V7.0 (TA Instruments Inc., New Castle, USA) equipped with an 18-N load cell as well as a tension film clamp. Films strips were stretched at a strain rate of 0.1% min-1 at 25 °C until failure. Tensile strength, Young’s modulus, and elongation at break of hybrid films were calculated. At least 5 specimens of each treatment were analyzed. Contact angle measurements were carried out on an optical contact angle meter (KSV Instruments, model CAM 101) in accordance with ASTM D5725-99(2008). Five microliters of ultrapure, deionized water were put in contact with the surfaces of films that had been conditioned to 50% RH as described previously. Images were acquired during 180 s by a digital camera (KSV Instruments, model CCD KSV-5000) and used for contact angle determinations in the KSV CAM2008 software. The procedure was performed at more than five spots per film. The thermal stability of films was investigated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA was performed on a Thermoanalyzer TG/DTA simultaneous SDT 2960 from TA Instruments, under the following conditions: alumina crucible, synthetic air (100 mL min−1), and heating at 10 oC min−1 from 30 to 600 oC. DSC was performed on about 5 mg of material within a sealed crucible on a TA2910 calorimeter (TA instruments) under flushing nitrogen (70 mL min−1) in the range of 0-360 oC and at a heating rate of 10 oC min−1. Indium and Zinc standards were used to perform the calibration of DSC measurements. Photoluminescence properties of free-standing hybrid films doped with YVO4:Eu3+ nanoparticles were investigated at room temperature. Excitation and emission spectra were recorded on a Horiba Jobin Yvon Fluorolog spectrofluorimeter model FL3-222, equipped with a Hamamatsu R928 photomultiplier and Xe lamp (450 W) using front face mode at a 0.5-nm resolution.

3.

Results and discussion



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Pristine SF and SFGPTMS hybrids films comprising different GPTMS contents were obtained by casting the aqueous mixture of SF and GPTMS onto polycarbonate dishes and then peeling the dried films off from the casting surface. The thicknesses of the free-standing films were very similar (~ 100 µm) for all compositions with standard deviations lower than 5% among the samples. Hybrid SFGPTMS films were insoluble in aqueous and conventional organic solvents and could reversibly bend in acute angles even at the lowest GPTMS content (i.e. 10 wt%). Besides that, all SF hybrid films were highly transparent in the visible region (transmittance values ranged from 79% to 90%), as shown in Figure 1.

Figure 1. (A) Transmittance of silk fibroin (SF)/(3-glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films (SFGPTMS) with different GPTMS contents (10 - 50 wt%) within the visible region. The inset shows a photography of a transparent and flexible SFGPTMS hybrid film (ca. 20 wt% of GPTMS) against a colorful background. (B) Refractive index of the hybrid films as a function of (3-glycidyloxypropyl)trimethoxysilane (GPTMS) content. The refractive index of hybrid films slight decreased from 1.5493 to 1.5254 as a function of the increase in GPTMS content (Figure 1B). The standard deviation of refractive index of samples


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was below 0.1% for all samples. From the high-accuracy prism coupling technique measurements, a quasi-linear relationship (with negative slope) between refractive index and GPTMS content in the hybrid films. It is evidenced that the decrease of refractive index arises from the incorporation of GPTMS into SF matrix. Interestingly, we observed that the transmittance of SFGPTMS hybrid films progressively decreased to a minimum of 79% at 550 nm as the GTPMS content was increased to 50 wt% (Figure 1). In order to get more insights about the influence of GPTMS on the surface morphology and optical properties, we conducted SEM and AFM analyses of SFGPTMS hybrid and pristine SF films, which are depicted in Figure 2 and Figure 3, respectively.



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Figure 2. Scanning electron microscopy images of (A and B) pristine SF films and silk fibroin (SF)/(3-glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films comprising (C and D) 10% (SFGPTMS 10%), (E and F) 30% (SFGPTMS 30%) or (G and H) 50% (SFGPTMS 50%) of GPTMS.


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Figure 3. Atomic-force microscopy (AFM) height images of (A) pristine SF films and silk fibroin (SF)/(3-glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films comprising (C) 10% (SFGPTMS 10%), (E) 30% (SFGPTMS 30%) or (G) 50% (SFGPTMS 50%) of GPTMS. AFM phase images of (B) pristine SF films and SF/GPTMS hybrid films containing (D) 10% (SFGPTMS 10%), (F) 30% (SFGPTMS 30%) or (H) 50% (SFGPTMS 50%) of GPTMS.



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The high magnification SEM images show a granular-like surface for SFGPTMS hybrid films with GPTMS content higher than 30 wt%. On the other hand, the SEM images of SFGPTMS 10% and pristine SF films (Figure 2) do not display such morphology. Interestingly, the increased GPTMS contents in SFGPTMS hybrid films made film surface smoother and more regular. This can be confirmed by the decreased surface roughness of the investigated films, which is strongly related to the height values of AFM images displayed in Figure 3 decreasing from 110 to 60 nm for pristine SF and SFGPTMS 50% films, respectively. The roughness (Rq) of the investigated SFGPTMS hybrid films extracted from the AFM height images confirms that the hybrid surface became smoother with increased GPTMS contents. The Rq changed from 15.7 ± 3.3 nm for pristine SF films to 6.9 ± 1.0 nm for SFGPTMS 50% films. The roughness values of the investigated SFGPTMS hybrid films are in agreement with the changes in AFM height images. Moreover, the contrast of the AFM phase images changed with increasing GPTMS contents, being only 22 º for pristine SF films and 83 º for SFGPTMS 50% films, leading to very regular surfaces. One may distinguish two different phases – one corresponding to SF-rich phase and the other corresponding to GPTMS-rich phase – indicating that hybrid surface was composed by two different materials. Phase AFM images of SFGPTMS hybrid films clearly revealed the presence of domain boundaries with remarkably differences in contrast level even if only 10% of GPTMS was added, implying that the hybrid surface has different composition. A more detailed investigation on the SF and GPTMS phases can be achieved using vibrational spectroscopy. Particularly, FTIR is a useful tool to study the conformational changes of SF. Amide I (1700-1600 cm−1) and amide II (1600-1500 cm−1) vibrational modes provide essential information about the secondary structures of the protein backbone. Figure 4 shows the FTIR spectra of pristine SF and SFGPTMS hybrid films.


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Figure 4. Fourier transform infrared spectra of (a) pristine SF films and silk fibroin (SF)/(3glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films containing (b) 10% (SFGPTMS 10%), (c) 30% (SFGPTMS 30%), (d) 50% (SFGPTMS 50%) and, (e) 70% (SFGPTMS 70%). The spectrum for pristine SF film was assigned to the Silk I conformation as evidenced by the position and intensity of the amide I (that is, C=O stretching vibrations), amide II (C–N stretching vibrations), and amide III (N–H deformation vibrations) bands at 1655, 1540, and 1235 cm−1, respectively. Indeed, characteristic Silk I vibrational modes can be distinguished by the weak bands at 1409 cm−1 for CαH2 stretching as well as 1387 cm−1 and 1330 cm−1 for CH3 stretching of alanine residue.36 On the other hand, Silk II was the predominant conformation after GPTMS addition. The typical broad band of amide III region at 1230 cm−1 for pristine SF film was assigned to Silk I structure. The relative intensity of this band was significantly reduced according with the



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GPTMS content. At the time, a prevailing band at 1256 cm−1 was found in the amide III region, which is assigned to epoxy ring breathing vibrational mode of GPTMS molecule coupled with βsheet conformation.36,37 The bands at 1106 cm−1 and the shoulder at 1130 cm−1 are assigned to the asymmetric stretching (Si-O-Si). The weak bands at 905 and 849 cm−1 indicate the hydrolysis of methoxyl groups with the formation of Si-OH groups and consequently an incomplete condensation of SiO-Si net.38 The role of silanol groups is the additional hydrogen interaction with the residual polar groups in SF backbone chain. The main N-H stretching absorption appeared at 3280 cm−1 (amide A region). The band got sharp and shifted as a function of GPTMS content. Indeed, the amide A region shifted ca. 12 cm−1 towards higher wavenumbers for SFGPTMS 50% and SFGPTMS 70% samples, which may be due to hydrogen interactions with GPTMS molecules, i.e. interactions between N-H bonds of protein backbone chain with Si-O polar groups or glycidoxy portion of GPTMS molecules. In addition, a shoulder band centered at 3265 cm−1 appeared after the introduction of GPTMS, band which is associated with -O-H stretching mode. The bands at 2936 and 2876 cm−1 can be clearly distinguished in the SFGPTMS hybrids being associated with CH2 asymmetric and symmetric stretching modes of glycidoxylpropyl backbone of GPTMS molecule, respectively.37 To complement the FTIR results, Raman spectroscopy was also carried out. The Raman spectra of pristine SF and SFGPTMS hybrids are shown in Figure 5. The Raman spectrum of pristine SF showed typical bands of Silk I conformation.39-41 The band at 1661 cm−1 is assigned to amide I vibration band of SF peptide chain.40 SF and SFGPTMS films showed almost the same bands in the amide I region. However, the amide I band underwent a slight shift to 1664 cm−1 and a narrowing in SFGPTMS samples, which is consistent with β-sheet structures produced from the transition of Silk I to Silk II conformations after the addition of GPTMS.39-41 The band at 1255 cm−1 and a component at 1236 cm−1 observed in pristine SF film are related to amide III from Silk I conformation. In addition, bands at 1100 cm−1 and a broad band at 938 cm−1 are assigned to C-C and C-N stretching, respectively, usually associated with some αhelices content from Silk I conformation.41 GPTMS addition caused meaningful changes in the amide III region. In SFGPTMS 10%, a shoulder emerged at 1230 cm−1, which was also observed for SF films rich in methanol-induced Silk II conformation.40 The strong band at 1256 cm−1


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observed for all SFGPTMS hybrids, regardless of the compositions, is assigned to C-H rock and C-C stretch vibration from epoxy ring of the GPTMS molecule.37

Figure 5. Raman spectra of silk fibroin (SF)/(3-glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films (a) 0% (pristine SF), (b) 10% (SFGPTMS 10%), (c) 30% (SFGPTMS 30%) and, (d) 50% (SFGPTMS 50%) of GPTMS. The modification of SF films with GPTMS is also confirmed by the presence of bands related to glycidoxypropyl backbone observed at 1030 and 1133 cm−1. The weak band at 1030 cm−1 is assigned to C-C stretch and C-O stretch of GPTMS propyl chain while the band at 1133 cm−1 is assigned to C-C tors, C-H (CH2) rock, and C-H rock vibration of glycidoxypropyl chain backbone coupled to the Si-O-Si/Si-OH stretching mode.42,43 Additionally, the Raman spectra of SFGPTMS films showed a new component at 1165 cm−1 assigned to CH2 rock vibration mode of propyl chain from GPTMS molecule. An additional band centered at 763 cm−1 also emerged as a function of GPTMS concentration being assigned to symmetric stretching of aliphatic ether linkage.37 The Raman spectra of SFGPTMS films also show that the condensation step was incomplete, which can be assessed by the strong and broad band at 1080 and 976 cm−1 regarding Si-O from siloxane bonds as well as from Si-OH stretching modes, respectively.44 It is important to note that



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it was not possible to observe any extra bonds between the siloxane groups and the SF protein from vibrational spectra of SFGPTMS films. We also analyzed pristine SF and SFGPTMS hybrid films by

13

C solid state NMR

spectroscopy. In general, the chemical shifts of alanine β-carbon (Cβ) are utilized to determine the conformation of the regenerated SF due to the variation of electron density generated owing to twist angle of the peptides. This assumption has been shown by 13C solid state NMR where the Cβ has increased shielding in the following order: β-sheet < random coil < α-helix.45 The antiparallel β-sheet (Silk II conformation) and the α-helix form of SF are well characterized based on conformation-dependence of the Cβ chemical shift of Gly, Ala, and Ser residues. Concerning GPTMS, the chemical environment of carbon nuclei observed by NMR is an important tool and can be used as a structural probe for observation of the hydrolysis/condensation process of silicon alkoxide and eventual organic network formation through polymerization of epoxy groups.

13 Figure 6. C-1H-MAS NMR spectra of films comprising different (3glycidyloxypropyl)trimethoxysilane (GPTMS) contents: (a) 0% (pristine SF film), (b) 10% (SFGPTMS 10%), (c) 30% (SFGPTMS 30%), (d) 50% (SFGPTMS 50%), (e) 70% (SFGPTMS 70%) and, (f) GPTMS powder. It is important to clarify that GPTMS itself did not result in freestanding films. The item (f) refers to the powder resulting from hydrolysis/condensation of GPTMS undertaken in the same experimental conditions utilized in the protocol of fabrication of SFGPTMS hybrids, except that SF was not introduced in the reaction mixture.


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Figure 6 shows the 13C-1H MAS NMR spectra of the pristine SF and SFGPTMS films prepared at different SF:GPTMS ratios as well as of GPTMS powder. GPTMS spectrum shows six resonance lines and their assignments were made on the basis of previous works46–48 and identified in the spectra of SFGPTMS 70%, SFGPTMS 50% and SFGPTMS 30% films. These are barely observed in SFGPTMS 10%. The peaks observed at 42.6 and 49.7 ppm (positions 6 and 5, respectively), corresponding to epoxy groups, appeared with the same intensity for all hybrid films, indicating that there was no polymerization process. These results are in agreement with vibrational spectroscopy data, i.e. unreacted epoxy ring from GPTMS molecule. The peak at 7.8 ppm is referred to the -CH2-SiO3/3 groups (position 1).48 The peak at 50 ppm is associated to O-CH3 (position 7) groups and was hardly identified in the spectra of SFGPTMS hybrids, which in turn is an indicative that siloxane groups were mostly hydrolyzed.46-48 Although we did not observe significant changes in the intensities and half-height width of the peaks, we observed increased magnetic shielding for all the peaks with the increase of SF content, suggesting the interaction between SF and GPTMS during formation of the SFGPTMS hybrid films. As already mentioned, the chemical shift of alanine Cβ is utilized to investigate the conformation of SF. The peak of alanine Cβ was observed as asymmetric and broad in the frequency range 20 to 0 ppm, showing contribution of two convoluted resonance lines, at 20.2 and 17.1 ppm. This suggests that the pristine SF film was formed by β-sheets (Silk II) and random coil conformations, respectively.49–51 Lines of alanine Cβ appear with decreasing intensity for the line at 17.7 ppm and increasing intensity of that at 20.2 ppm as a function of GPTMS content from 10 to 70 wt%. Clearly, the addition of GPTMS favors the formation of hybrid films with Silk II conformation whereas pristine SF exhibits random conformation. The aforementioned results emphasize that most epoxy rings in SFGPTMS hybrids are available to be further modified. The epoxy groups provide a new pathway to chemically modify the SF films. Through ring-opening reactions it is possible to obtain a cross-linked hybrid matrix, which in turn can be conducted by nucleophile substitution under alkaline medium or by exposition to ultraviolet light. XRD patterns are shown in Figure 7. Considering the as-cast pristine SF film, a weak diffraction peak at 2θ= 11.8 º and a broad halo peak at 22.5 º corresponding to 7.5 and 3.9 Å are observed. Pristine SF film displays mostly amorphous or Silk I conformation, which is in accordance to previous studies.14,52



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After the addition of 20 wt% of GPTMS, the d-spacing of 3.9 Å disappeared and peaks at 2θ= 12 º (weak), 20.5 º (moderate), and 24.5 º (weak) corresponding to 7.4, 4.3 and 3.6 Å became gradually sharper. These peaks clearly indicate that Silk I conformation found for pristine SF films was mostly converted to crystalline Silk II conformation16 upon the addition of at least 20 wt% of GPTMS. The d-spacing at 4.3 Å (2θ= 20.5 º) is attributed to intra-hydrogen-bonded βsheet spacing.14 Beyond 30 wt% of GPTMS a new peak was observed at 6.3 º and became predominant together with that at 20.5 º.

Figure 7. X-ray diffraction patterns of (a) pristine silk fibroin (SF) film and SF films added by (b) 10% (SFGPTMS 10%), (c) 20% (SFGPTMS 20%), (d) 30% (SFGPTMS 30%), and (e) 50% (SFGPTMS 50%) of (3-glycidyloxypropyl)trimethoxysilane (GPTMS). The peak emerging at 6.3 ° in SFGPTMS hybrids has been attributed to inter-particle scattering interference.53 This peak indicates highly non-periodic fluctuation of the electronic density in the SFGPTMS hybrid films, which is eventually observed due to silica aggregation at high silylating agent concentrations.53 Once a critical GPTMS concentration is achieved, it is reasonable to expect that the connection between Si-O- sites would overcome the steric hindrance imposed by ACS Paragon Plus Environment

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SF polymeric chain and thus promote the formation of siloxane-rich domains as result of the hydrolysis/condensation reactions. The average inter-domains distance is estimated to be 13-14 Å and support the development of granular surface observed in SEM images and the enhancement of height uniformity among the domain boundaries observed in AFM data. Noteworthy vibrational, NMR spectroscopies as well as diffractometry results confirm that SFGPTMS hybrid films present high content of Silk II conformation. On the course of the drying of the films, shear forces could induce a partial crystallization of Silk I to Silk II conformation. As illustrated in Figure 8, it is feasible that GPTMS molecules undergo hydrolysis and condensation reactions during the preparation of hybrid films leading to silica networks or particles when mixed with an aqueous SF solution.

Figure 8. Schematic representation demonstrating the changes of conformation of silk fibroin (SF) films as a function of (3-glycidyloxypropyl)trimethoxysilane (GPTMS). With the increase of GPTMS content, the amorphous Silk I conformation composed mainly by α-helix conformation is predominantly converted to crystalline Silk II conformation which is mostly composed by β-sheet secondary structures. In addition, SFGPTMS films have large amount of epoxy rings and, eventually, silanol groups available for further functionalization according with GPTMS content, thereby extending the chemical reactivity of hybrids films based on SF. It is important to highlight that methanol is a byproduct formed in situ from GPTMS hydrolysis and may play an important role on the conformation changes observed in the SFGPTMS hybrids since it is a well-known solvent used to induce the conversion of SF amorphous Silk I to crystalline Silk II.16 Methanol is a hydrophilic agent with high dehydrating property. Methanol



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generated during the fabrication of the SFGPTMS hybrid films could rapidly induce the crystallization of SF by increasing its gelation rate. As evidenced from the results aforementioned, this process leads to structural transition from Silk I to Silk II conformation. Moreover, we suggest that at intermediate concentrations of GPTMS (i.e. 30 - 50 wt%), the concentration of GPTMS and, therefore the concentration of generated methanol would be high enough to promote the segregation of SF- and GPTMS-rich domains mostly caused by dehydration process on the protein backbone. The slight decrease of the transparency in the hybrids comprising high content of GPTMS could be addressed to the increase in the number of micro-sized aggregates that scatter the light. Most significantly, the hybrid films deliver flexible transparent films rich in epoxy (organic) and silanol (inorganic, e.g. from incomplete condensation of silica network) groups available for further functionalization. Obviously, the number of those reactive functional groups depends on GPTMS content. The thermal stability of the films has been investigated by thermogravimetry (TGA) and differential scanning calorimetry (DSC). Pristine SF film has a well reported thermogravimetric curve54 that can be divided in three subsequent regions (regions I, II, and III), characterized by evidently different mass loss rates. Region I (35-40 to 117-170 ºC) is related to water loss. Regions II (170 to 240 ºC), and III (> 240 oC) provided evidence of SF degradation. Above 200 ºC, side chain groups of amino acid residues of silk backbone started to degrade producing lowweight gas molecules such as CO, CO2, and NH3.55 At this temperature range, epoxy groups undertake thermal decomposition through homolytic scission of chemical bonds and simultaneous dehydration of water molecules from oxypropylene.56 As shown in Figure 9, the mass loss in region I gradually decreased with the increase of the GPTMS content. The mass loss was 10% for pristine SF and 4% for SFGPTMS 50%. The decomposition temperature of SF in region III changed to higher values with increasing the GPTMS content. The results could indicate a conformation change of SF to its β-sheet form after adding the GPTMS, which is in good agreement with the aforementioned results. As indicated in Figure 9, these characteristics could be related to the higher stability against thermal decomposition induced by β-sheet formation. The same characteristics were observed by Motta et al.54 after inducing the crystallization (Silk II conformation) of SF films by treatment with ethanol.


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Figure 9. Thermogravimetric curves of SF/(3-glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films (SFGPTMS). DSC curves for pristine SF and SFGPTMS films are shown in Figure 10. The DSC profile of pristine SF film displayed three major events. The first endothermic peak centered at about 82 oC has been attributed to the loss of adsorbed water.57 The higher temperature of the endothermic peak (284 ºC) could be attributed to the to the thermal decomposition of SF chains. Thermal decomposition was also confirmed by TGA curves. Pristine SF film exhibited an endothermic peak attributed to thermal degradation event at 283 °C. Thermal degradation peaks of SF films at temperatures below 290 °C are characteristic of amorphous Silk I conformation and were present when SF films had not been not submitted to any kind of physical or chemical treatment to induce its conformation to a more stable and crystalline Silk II conformation.58 The exothermic peak at 218 °C is attributed to the crystallization process from Silk I to Silk II conformation.58



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Figure 10. DSC curves of (a) pristine silk fibroin (SF) and SF/(3glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films comprising (b) 20% (SFGPTMS 20%), (c) 30% (SFGPTMS 30%) and, (d) 50% (SFGPTMS 50%) of GPTMS. The DSC curves of all SFGPTMS films evidence important changes in relation to the DSC curve of pristine SF film. For SFGPTMS films, the first endothermic peak also corresponds to the contribution of elimination of water molecules during silica condensation. Once the introduction of GPTMS into the hybrid composition induces the conversion of Silk I to crystalline Silk II conformation, the DSC curves of SFGPTMS hybrids do not show exothermic peak. When GPTMS concentration is increased, the endothermic peak found at 284 ºC for pristine SF shifted to higher temperatures: 296 ºC for SFGPTMS 30% and 315 ºC for SFGPTMS 50%. This behavior was also observed in the TGA curves. The sharp endothermic event is due to thermal degradation of the native protein. The SFGPTMS film containing 50 wt% of GPTMS clearly shows two subsequent peaks within this temperature range. The exothermic event is attributed to GPTMS degradation mostly described by polymerization of epoxy ring and silica condensation reactions. Evidently, the intensity of SF degradation peak decreases with increasing GPTMS contents. The shift of the degradation temperature can be attributed to the interaction between the SF and GPTMS as previously discussed by vibrational and NMR spectroscopies.


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Mechanical assays provide valuable information regarding the inherent behavior of materials in response to tension and deformation. Tensile properties are the most investigated mechanical properties in free-standing films. Moderate tensile strength, low elongation at break, and high Young's modulus values are typical of rigid, brittle films, whereas moderate tensile strength, high elongation at break, and low Young's modulus values suggest that the material is soft and tough. Rigid and tough films, in turn, are characterized by high elongation at break, Young's modulus, and tensile strength values. Figure 11A evidences that both tensile strength and Young’s modulus of pristine SF films were higher than those of hybrid films, suggesting that GPTMS played a plasticizing role in by reducing film stiffness from brittleness. This behavior is typical of low molecular weight molecules (e.g. GPTMS; Mw= 236 g mol−1), which are believed to imbed in the free volume among adjacent polymer chains in a way that the intermolecular distance and chain mobility are increased, therefore weakening the overall intermolecular interactions that are responsible for film resistance and rigidity.59 The schematic representation proposed in Figure 8 also illustrates the positioning of GPTMS within SF chains. It is clear that GPTMS addition affected tensile strength, but GPTMS content appears not to play a significant role since this mechanical attribute tented to assume a plateau after the addition of the lowest assessed GPTMS content. This behavior somehow differed from that of Young’s modulus, which in turn was remarkably dependent on GPTMS concentration, as indicated by the linear model fitted to the data. Although both mechanical attributes are highly sensitive to the action of plasticizers, this difference could be explained by the fact that Young’s modulus is measured at the very beginning of the stress vs. strain curve, i.e. when the viscoelastic regime is still linear. Tensile strength, on the other hand, denotes the ultimate strength that the specimen can stand prior to failure and is measured at higher deformations, when polymer chains are highly stretched and the occurrence of physical entanglements is remarkably lower.60 Interestingly, elongation at break was not affected by GPTMS addition (p= 0.391 > 0.05).



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Figure 11. (A) Tensile strength, Young’s modulus and elongation at break of silk fibroin (SF)/(3glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films as a function of GPTMS content. (B) Contact angle of silk fibroin (SF)/(3-glycidyloxypropyl)trimethoxysilane (GPTMS) hybrid films (SFGPTMS) as a function of GPTMS content: 0% (pristine SF), 10%, 30%, and 50%. The insets are images of water droplets deposited onto pristine SF and SFGPTMS 50% films for 180 s. Figure 11B shows typical contact angles between the films and water droplets. Contact angle indicates the affinity between the liquid and the film surface, higher values indicating poorer affinities. The contact angle of the pristine SF film was reduced throughout time and was shown to be remarkably lower than those of the hybrid films. This information is equivalent to state that GPTMS-free film absorbed water to a higher extent than its GPTMS-containing counterparts – which is evidenced by the droplet images in the insets of Figure 11B – as well as that the former is more hydrophilic than the latters. This is attributed to the α-helix-to-β-sheet transition upon GPTMS addition, which is in close agreement with the outcomes of previously discussed techniques. Unlike α-helices of Silk I, β-sheets (anti-parallel crystal conformation) of Silk II serve as physical cross-links that render SF films insoluble in water.52 Silk I, in turn, has been reported to be soluble in water.61 Indeed, such transition has been reported to increase the water insolubility of SF films.14-16 Although a gradual transition from α-helix to β-sheet has been proposed (Figure 9), GPTMS content appears not to affect the hydrophobicity of SF films. Preliminary results of cytotoxicity/viability assays for osteoblasts cells clearly indicate that there was no significant statistical difference (p≥0.05) between cell viability of pristine SF and SFGPTMS hybrid films, regardless of GTPMS concentration as shown in Figure 12.


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Figure 12. MTT-formazan viability/cytotoxicity assay of pristine SF (control) and SFGPTMS hybrid films performed with osteoblasts lineage derived from calvaria of neonatal rats (OSTEO1). Results are expressed as percentage of cell viability relative to SF membranes. ANOVA ONE WAY: p=0.244. Our study found that SFGPTMS films were non-cytotoxic for osteoblasts cells. Since SF is a well-known biocompatible natural polymer,62–64 it was expected that SF-rich hybrid films would preserve this feature. On the other hand, we also evaluated the cytotoxicity of hybrids films composed of GPTMS as major component in weight in order to identify the capabilities of SFGPTMS hybrids for biophotonics. Noteworthy, we identified that hybrids containing up to 30 wt% of GPTMS (i.e. 70% of SF or SFGPTMS 30%) and films with opposite composition (i.e. 70 wt% of GPTMS, 30 wt% of SF or SFGPTMS 70%) had negligible toxicity comparing with control. The absence of cytotoxicity for hybrids rich in epoxy groups has been also verified by recent studies involving the preparation of GPTMS-based organic-inorganic polymer nanocomposites.65-67 Remarkably, this feature enable us to further explore SFGPTMS hybrid films as potential functional biomaterials. Nevertheless, additional biological assays are in progress to gather a wider range of medical applications.



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Figure 13. (A) Excitation (λem = 620 nm) and emission (λexc = 303 nm) spectra of an aqueous suspension of as-prepared YVO4:Eu3+ nanoparticles. The YVO4:Eu3+ nanoparticles were roughly spherical and smaller than 5 nm. The inset shows the high-resolution electron microscopy image of YVO4:Eu3+ nanoparticles. (B) Excitation and emission spectra of SFGPTMS 20% film containing 5 wt% of YVO4:Eu3+ nanoparticles. The excitation spectrum was performed by monitoring at 620 nm while the emission measurement was performed under excitation of 303 nm. The inset shows the amplified region highlighted in dashed red line of excitation spectrum for clearer observation. In order to evaluate the SFGPTMS hybrids films for photonics, luminescent films comprising transparent SFGPTMS 20% hybrid loaded with 5 wt% YVO4:Eu3+ nanoparticles were prepared. The excitation and emission spectra of an aqueous suspension of YVO4:Eu3+ nanoparticles are shown in Figure 13A. The excitation and emission spectra of the luminescent hybrid are shown in Figure 13B. The HRTEM image of Figure 13A inset shows that the non-spherical YVO4:Eu3+


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particles were smaller than 5 nm. From the excitation spectrum of YVO4:Eu3+ nanoparticles and luminescent SFGPTMS hybrid, a broad band around 300 nm is typically attributed to the charge transfer from the oxygen ligands to the central vanadium atom.68 In both excitation spectra of YVO4:Eu3+ nanoparticles and luminescent hybrid film, the characteristic transitions within Eu3+ 4f6 configuration are detected at 396 and 467 nm with a very weak intensity attributed to 7F0–5L6 and 7F0–5D2 transitions. A strong and broad emission band with small Stokes shift (ca. 30 nm) was observed at 334 nm being mainly attributed to fluorescence of aromatic residues of SF (i.e. tyrosine and tryptophan) together with radiative recombination of SiO2 matrix produced in SFGPTMS hybrids. Narrower bands peaking in the range of 570–710 nm region in the emission spectrum of aqueous suspension of YVO4:Eu3+ shown in Figure 13B is clearly detected in the emission spectrum of SFGPTMS hybrid. These bands were assigned to Eu3+ 4f6 transitions arising from the 5D0 excited state to the 7FJ manifold (J= 0, 1, 2, 3, and 4). Once Eu3+ ions are located at low symmetric local sites in the YVO4 host lattice, the most intense emission band of Eu3+ concerns the hypersensitive transition 5D0- 7F2 at about 620 nm. In the emission spectrum of both samples not only the characteristic transition lines from the lowest excited 5D0 level of Eu3+ are observed, but also those from higher energy levels such as 5D1-7F1 with a very weak intensity. When the spectra of the luminescent SFGPTMS hybrid and YVO4:Eu3+ nanoparticles are compared, it is evident that the resolution is better in the case of pristine nanoparticles. The loss of resolution in the hybrid matrix can be explained by site-to-site inhomogeneity caused by differences in the outer coordination sphere of Eu3+ resulting in small variations in the crystalfield strength felt by the different Eu3+ ions. As previously reported, the Eu3+ emission could be excited through efficient antenna effect involving vanadate charge transfer states in resonance with excited states of SF aromatic amino acids (i.e. tyrosine and tryptophan) present in the medium.31

4. Conclusion In summary, we report the fabrication of flexible and insoluble free-standing SF films rich in epoxy and silanol functional groups with high transparency (ca. 90 %) even at high GPTMS contents (up to 70 wt%) by using all-aqueous sol-gel approach. We showed that hybrid films with remarkable optical quality were achieved at GPTMS concentration higher than 60 wt% or lower than 20 wt% whereas the refractive index displayed a quasi-linear dependence on GPTMS



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content. This method allowed us to achieve SF hybrid films with relatively smoother surface morphology upon the addition of GPTMS whose roughness decreased to less than half value compared to pristine SF film. We carefully evaluated the conformation changes of SF from amorphous Silk I to crystalline Silk II in response the introduction of GPTMS by FTIR, Raman, and

13

C NMR spectroscopies as well XRD measurements. This transition rendered hydrophobic

hybrid films. None of the investigated hybrid films with overall composition ranging from 10 to 70 wt% of GPTMS content showed cytotoxicity to osteoblastic cells. Taking advantage of their remarkable optical properties, we demonstrated the feasibility to tailor functional and luminescent SF hybrid films by loading red emissive 5 nm YVO4:Eu3+ nanoparticles. Optically clear, flexible and biocompatible epoxy-rich silk fibroin substrates are particularly attractive supports for potential immobilization of (bio)molecules through epoxy-ring opening reactions or via silanol condensation. Furthermore, hybrid SF films are versatile matrix that may potentially host a myriad of luminescent probes such as lanthanide-based compounds, organic dyes and carbon/noble-metal/quantum dots which can be useful for the visualization of optically tagged molecules in regenerative tissue engineering and for fluorescence sensing based on analyteinduced luminescence switching.

Acknowledgment The authors are grateful to the financial support from the Brazilian agencies: Coordenacão de Pessoal de Ensino Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientfico e Tecnológico (CNPq). We also acknowledge the National Laboratory of Nanotechnology (LME/LNNano/CNPEM) and Dr. Além-Mar B. Gonçalves for the technical support during the microscopy data collection. R. R. Silva thanks to the Fundacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the Grant No. 2013/12367-6.

References (1)

Prasad, P. N. Biomaterials for Photonics. In Introduction to Biophotonics; John Wiley & Sons: Hoboken, 2003; p 545–572.

(2)

Lawrence, B. D.; Omenetto, F.; Chui, K.; Kaplan, D. L. Processing Methods to Control Silk Fibroin Film Biomaterial Features. J. Mater. Sci. 2008, 43, 6967–6985.


Page 31 of 37

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(3)

Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Silk-Based Biomaterials. Biomaterials 2003, 24, 401–416.

(4)

Omenetto, F. G.; Kaplan, D. L. A New Route for Silk. Nat. Photonics 2008, 2, 641–643.

(5)

Sashina, E. S.; Bochek, A. M.; Novoselov, N. P.; Kirichenko, D. A. Structure and Solubility of Natural Silk Fibroin. Russ. J. Appl. Chem. 2006, 79, 869–876.

(6)

Amsden, J. J.; Domachuk, P.; Gopinath, A.; White, R. D.; Negro, L. D.; Kaplan, D. L.; Omenetto, F. G. Rapid Nanoimprinting of Silk Fibroin Films for Biophotonic Applications. Adv. Mater. 2010, 22, 1746–1749.

(7)

Silva, R. R.; Dominguez, C. T.; dos Santos, M. V.; Barbosa-Silva, R.; Cavicchioli, M.; Christovan, L. M.; de Melo, L. S. A.; Gomes, A. S. L.; de Araújo, C. B.; Ribeiro, S. J. L. Silk Fibroin Biopolymer Films as Efficient Hosts for DFB Laser Operation. J. Mater. Chem. C 2013, 1, 7181.

(8)

Zhou, C. Z.; Confalonieri, F.; Jacquet, M.; Perasso, R.; Li, Z. G.; Janin, J. Silk Fibroin: Structural Implications of a Remarkable Amino Acid Sequence. Proteins 2001, 44, 119– 122.

(9)

Kim, D.-H.; Viventi, J.; Amsden, J. J.; Xiao, J.; Vigeland, L.; Kim, Y.-S.; Blanco, J. A.; Panilaitis, B.; Frechette, E. S.; Contreras, D.; Kaplan, D. L.; Omenetto, F. G.; Huang, Y.; Hwang, K.-C.; Zakin, M. R.; Litt, B.; Rogers, J. A. Dissolvable Films of Silk Fibroin for Ultrathin Conformal Bio-Integrated Electronics. Nat. Mater. 2010, 9, 511–517.

(10)

Perry, H.; Gopinath, A.; Kaplan, D. L.; Dal Negro, L.; Omenetto, F. G. Nano- and Micropatterning of Optically Transparent, Mechanically Robust, Biocompatible Silk Fibroin Films. Adv. Mater. 2008, 20, 3070–3072.

(11)

Harris, M.; Johnson, T. B. Study of the Fibroin from Silk in the Isoelectric Region 1. Ind. Eng. Chem. 1930, 22, 539–542.

(12) Bhat, N. V.; Ahirrao, S. M. Investigation of the Structure of Silk Film Regenerated with Lithium Thiocyanate Solution. J. Polym. Sci. Polym. Chem. Ed. 1983, 21, 1273–1280. (13)

Goujon, N.; Wang, X.; Rajkowa, R.; Byrne, N. Regenerated Silk Fibroin Using Protic Ionic Liquids Solvents: Towards an All-Ionic-Liquid Process for Producing Silk with Tunable Properties. Chem. Commun. 2012, 48, 1278–1280.

(14)

Jin, H.-J.; Park, J.; Karageorgiou, V.; Kim, U.-J.; Valluzzi, R.; Cebe, P.; Kaplan, D. L. Water-Stable Silk Films with Reduced β-Sheet Content. Adv. Funct. Mater. 2005, 15, 1241–1247.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(15)

Page 32 of 37

Hu, X.; Shmelev, K.; Sun, L.; Gil, E.-S.; Park, S.-H.; Cebe, P.; Kaplan, D. L. Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing. Biomacromolecules 2011, 12, 1686–1696.

(16) Magoshi, J.; Magoshi, Y.; Nakamura, S. Physical Properties and Structure of Silk. VII. Crystallization of Amorphous Silk Fibroin Induced by Immersion in Methanol. J. Polym. Sci. Polym. Phys. Ed. 1981, 19, 185–186. (17) Lu, S.; Wang, X.; Lu, Q.; Zhang, X.; Kluge, J. a.; Uppal, N.; Omenetto, F.; Kaplan, D. L. Insoluble and Flexible Silk Films Containing Glycerol. Biomacromolecules 2010, 11, 143– 150. (18)

Dang, Q.; Lu, S.; Yu, S.; Sun, P.; Yuan, Z. Silk Fibroin/montmorillonite Nanocomposites: Effect of pH on the Conformational Transition and Clay Dispersion. Biomacromolecules 2010, 11, 1796–1801.

(19)

Noishiki, Y.; Nishiyama, Y.; Wada, M.; Kuga, S.; Magoshi, J. Mechanical Properties of Silk Fibroin-Microcrystalline Cellulose Composite Films. J. Appl. Polym. Sci. 2002, 86, 3425–3429.

(20) Wang, Y. X.; Qin, Y. P.; Kong, Z. J.; Wang, Y. J.; Ma, L. Glutaraldehyde Cross-Linked Silk Fibroin Films for Controlled Release. Adv. Mater. Res. 2014, 887-888, 541–546. (21)

Jung, R.; Kim, H. S.; Kim, Y.; Kwon, S. M.; Lee, H. S.; In, H. J. Electrically Conductive Transparent Papers Using Multiwalled Carbon Nanotubes. J. Polym. Sci. Part B-Polymer Phys. 2008, 46, 1235–1242.

(22) Chen, T.; Embree, H. D.; Wu, L.-Q.; Payne, G. F. In Vitro Protein-Polysaccharide Conjugation: Tyrosinase-Catalyzed Conjugation of Gelatin and Chitosan. Biopolymers 2002, 64, 292–302. (23) Gotoh, Y.; Tsukada, M.; Minoura, N. Chemical Modification of Silk Fibroin with Cyanuric Chloride-Activated Poly(ethylene Glycol): Analyses of Reaction Site by Proton NMR Spectroscopy and Conformation of the Conjugates. Bioconjug. Chem. 1993, 4, 554– 559. (24) Tamada, Y. Sulfation of Silk Fibroin by Chlorosulfonic Acid and the Anticoagulant Activity. Biomaterials 2004, 25, 377–383. (25) Murphy, A. R.; John, P. St.; Kaplan, D. L. Modification of Silk Fibroin Using Diazonium Coupling Chemistry and the Effects on hMSC Proliferation and Differentiation. Biomaterials 2008, 29, 2829–2838. (26) Sagnella, A.; Zambianchi, M.; Durso, M.; Posati, T.; Del Rio, A.; Donnadio, A.; Mazzanti, A.; Pistone, A.; Ruani, G.; Zamboni, R.; Benfenati, V.; Melucci, M. APTES Mediated Modular Modification of Regenerated Silk Fibroin in a Water Solution. RSC Adv. 2015, 5,


Page 33 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


63401–63406. (27) Innocenzi, P.; Martucci, A.; Guglielmi, M.; Armelao, L.; Pelli, S.; Righini, G. .; Battaglin, G. . Optical and Surface Properties of Inorganic and Hybrid Organic–inorganic Silica– titania Sol–gel Planar Waveguides. J. Non. Cryst. Solids 1999, 259, 182–190. (28)

Ribeiro, S. J. L.; Messaddeq, Y.; Gonçalves, R. R.; Ferrari, M.; Montagna, M.; Aegerter, M. A. Low Optical Loss Planar Waveguides Prepared in an Organic–inorganic Hybrid System. Appl. Phys. Lett. 2000, 77, 3502.

(29) Innocenzi, P.; Brusatin, G.; Guglielmi, M.; Signorini, R.; Bozio, R.; Maggini, M. 3(Glycidoxypropyl)-trimethoxysilane–TiO2 Hybrid Organic–inorganic Materials for Optical Limiting. J. Non. Cryst. Solids 2000, 265, 68–74. (30) Brusatin, G.; Abbotto, A.; Beverina, L.; Pagani, G. A.; Casalboni, M.; Sarcinelli, F.; Innocenzi, P. Poled Sol-Gel Materials With Heterocycle Push-Pull Chromophores That Confer Enhanced Second-Order Optical Nonlinearity. Adv. Funct. Mater. 2004, 14, 1160– 1166. (31) Lima, L. R.; Moraes, M. L.; Nigoghossian, K.; Peres, M. F. S.; Ribeiro, S. J. L. Silk Fibroin-Antigenic Peptides-YVO4:Eu3+ Nanostructured Thin Films as Sensors for Hepatitis C. J. Lumin. 2016, 170, 375–379. (32) Kamra, T.; Chaudhary, S.; Xu, C.; Johansson, N.; Montelius, L.; Schnadt, J.; Ye, L. Covalent Immobilization of Molecularly Imprinted Polymer Nanoparticles Using an Epoxy Silane. J. Colloid Interface Sci. 2015, 445, 277–284. (33) Mateo, C.; Grazu, V.; Palomo, J. M.; Lopez-Gallego, F.; Fernandez-Lafuente, R.; Guisan, J. M. Immobilization of Enzymes on Heterofunctional Epoxy Supports. Nat. Protoc. 2007, 2, 1022–1033. (34) Mitropoulos, A. N.; Marelli, B.; Ghezzi, C. E.; Applegate, M. B.; Partlow, B. P.; Kaplan, D. L.; Omenetto, F. G. Transparent, Nanostructured Silk Fibroin Hydrogels with Tunable Mechanical Properties. ACS Biomater. Sci. Eng. 2015, 1, 964–970. (35) Guan, W.; Zhou, W.; Lu, J.; Lu, C. Luminescent Films for Chemo- and Biosensing. Chem. Soc. Rev. 2015, 44, 6981–7009. (36)

Barker, M. G.; Hooper, A. J. Preparation and X-Ray Powder Diffraction Patterns of the Sodium Vanadates NaVO3, Na4V2O7, and Na3VO4. J. Chem. Soc. Dalt. Trans. 1973, 0, 1513-1517.

(35)

Giaume, D.; Buissette, V.; Lahlil, K.; Gacoin, T.; Boilot, J.-P.; Casanova, D.; Beaurepaire, E.; Sauviat, M.-P.; Alexandrou, A. Emission Properties and Applications of Nanostructured Luminescent Oxide Nanoparticles. Prog. Solid State Chem. 2005, 33, 99– 106.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 37

(36)

Taddei, P.; Monti, P. Vibrational Infrared Conformational Studies of Model Peptides Representing the Semicrystalline Domains of Bombyx Mori Silk Fibroin. Biopolymers 2005, 78, 249–258.

(37)

Sapić, I. M.; Bistricić, L.; Volovsek, V.; Dananić, V.; Furić, K. DFT Study of Molecular Structure and Vibrations of 3-Glycidyloxypropyl trimethoxysilane. Spectrochim. Acta. A. Mol. Biomol. Spectrosc. 2009, 72, 833–840.

(38)

Almeida, R. M.; Pantano, C. G. Structural Investigation of Silica Gel Films by Infrared Spectroscopy. J. Appl. Phys. 1990, 68, 4225.

(39)

Shao, J.; Zheng, J.; Liu, J.; Carr, C. M. Fourier Transform Raman and Fourier Transform Infrared Spectroscopy Studies of Silk Fibroin. J. Appl. Polym. Sci. 2005, 96, 1999–2004.

(40)

Monti, P.; Freddi, G.; Bertoluzza, A.; Kasai, N.; Tsukada, M. Raman Spectroscopic Studies of Silk Fibroin from Bombyx Mori. J. Raman Spectrosc. 1998, 29, 297–304.

(41)

Zheng, S.; Li, G.; Yao, W.; Yu, T. Raman Spectroscopic Investigation of the Denaturation Process of Silk Fibroin. Appl. Spectrosc. 1989, 43, 1269–1272.

(42)

Liu, H.; Zheng, S.; Nie, K. Morphology and Thermomechanical Properties of Organic−Inorganic Hybrid Composites Involving Epoxy Resin and an Incompletely Condensed Polyhedral Oligomeric Silsesquioxane. Macromolecules 2005, 38, 5088–5097.

(43)

Riegel, B.; Blittersdorf, S.; Kiefer, W.; Hofacker, S.; Müller, M.; Schottner, G. Kinetic Investigations of Hydrolysis and Condensation of the Glycidoxypropyltrimethoxysilane/aminopropyltriethoxy-Silane System by Means of FTRaman Spectroscopy I. J. Non. Cryst. Solids 1998, 226, 76–84.

(44) Klein, L. C.; Kordas, G. Electron-Spin Resonance and Other Spectroscopies Used in Characterizing Sol-Gel Processing. MRS Proc. 1986, 73, 461. (45) Kaplan, D. L.; Mello, S. M.; Arcidiacono, S.; Fossey, S.; Senecal, K.; Muller, W. Silk. In Protein based materials; Kaplan, D. L.; McGrath, K., Ed.; Birkhauser: Boston, 1998; p 103. (46) Alonso, B.; Massiot, D.; Babonneau, F.; Brusatin, G.; Giustina, G. Della; Kidchob, T.; Innocenzi, P. Structural Control in Germania Hybrid Organic−inorganic Materials. Chem. Mater. 2005, 17, 3172–3180. (47)

Brus, J.; Hlavatá, D.; Strachota, A. Self-Organization, Structure, Dynamic Properties, and Surface Morphology of Silica/Epoxy Films As Seen by Solid-State NMR, SAXS, and AFM. Macromolecules 2004, 37, 1346–1357.

(48)

Innocenzi, P.; Brusatin, G.; Babonneau, F. Competitive Polymerization between Organic and Inorganic Networks in Hybrid Materials. Chem. Mater. 2000, 12, 3726–3732.


Page 35 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(49)

Asakura, T.; Yao, J.; Yamane, T.; Umemura, K.; Ulrich, A. S. Heterogeneous Structure of Silk Fibers from Bombyx M Ori Resolved by 13C Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 2002, 124, 8794–8795.

(50)

Asakura, T.; Nakazawa, Y.; Ohnishi, E.; Moro, F. Evidence from 13 C Solid-State NMR Spectroscopy for a Lamella Structure in an Alanine-Glycine Copolypeptide: A Model for the Crystalline Domain of Bombyx Mori Silk Fiber. Protein Sci. 2005, 14, 2654–2657.

(51)

Zhou, P.; Li, G.; Shao, Z.; Pan, X.; Yu, T. Structure of Bombyx Mori Silk Fibroin Based on the DFT Chemical Shift Calculation. J. Phys. Chem. B 2001, 105, 12469–12476.

(52)

Lu, Q.; Hu, X.; Wang, X.; Kluge, J. A.; Lu, S.; Cebe, P.; Kaplan, D. L. Water-Insoluble Silk Films with Silk I Structure. Acta Biomater. 2010, 6, 1380–1387.

(53)

Xi, F.; Wu, J.; Lin, X. Novel Nylon-Supported Organic–inorganic Hybrid Membrane with Hierarchical Pores as a Potential Immobilized Metal Affinity Adsorbent. J. Chromatogr. A 2006, 1125, 38–51.

(54)

Motta, A.; Fambri, L.; Migliaresi, C. Regenerated Silk Fibroin Films: Thermal and Dynamic Mechanical Analysis. Macromol. Chem. Phys. 2002, 203, 1658–1665.

(55)

Schoeser, M. The Science of Silk. In Silk; Yale University Press: New Haven, 2007; p 234.

(56)

Macan, J.; Brnardić, I.; Orlić, S.; Ivanković, H.; Ivanković, M. Thermal Degradation of Epoxy–silica Organic–inorganic Hybrid Materials. Polym. Degrad. Stab. 2006, 91, 122– 127.

(57)

Magoshi, J.; Magoshi, Y.; Nakamura, S.; Kasai, N.; Kakudo, M. Physical Properties and Structure of Silk. V. Thermal Behavior of Silk Fibroin in the Random-Coil Conformation. J. Polym. Sci. Polym. Phys. Ed. 1977, 15, 1675–1683.

(58)

Moraes, M. A. de; Nogueira, G. M.; Weska, R. F.; Beppu, M. M. Preparation and Characterization of Insoluble Silk Fibroin/chitosan Blend Films. Polymers. 2010, 2, 719– 727.

(59)

Otoni, C. G.; Avena-Bustillos, R. J.; Olsen, C. W.; Bilbao-Sáinz, C.; McHugh, T. H. Mechanical and Water Barrier Properties of Isolated Soy Protein Composite Edible Films as Affected by Carvacrol and Cinnamaldehyde Micro and Nanoemulsions. Food Hydrocoll. 2016, 57, 72–79.

(60)

Pola, C. C.; Medeiros, E. A. A.; Pereira, O. L.; Souza, V. G. L.; Otoni, C. G.; Camilloto, G. P.; Soares, N. F. F. Cellulose Acetate Active Films Incorporated with Oregano (Origanum Vulgare) Essential Oil and Organophilic Montmorillonite Clay Control the Growth of Phytopathogenic Fungi. Food Packag. Shelf Life, 2016, 9, 69–78.

(61) Wilson, D.; Valluzzi, R.; Kaplan, D. Conformational Transitions in Model Silk Peptides.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 37

Biophys. J. 2000, 78, 2690–2701. (62)

Yang, Y.; Chen, X.; Ding, F.; Zhang, P.; Liu, J.; Gu, X. Biocompatibility Evaluation of Silk Fibroin with Peripheral Nerve Tissues and Cells in Vitro. Biomaterials 2007, 28 (9), 1643–1652.

(63)

Tang, X.; Ding, F.; Yang, Y.; Hu, N.; Wu, H.; Gu, X. Evaluation on in Vitro Biocompatibility of Silk Fibroin-Based Biomaterials with Primarily Cultured Hippocampal Neurons. J. Biomed. Mater. Res. Part A 2009, 91A, 166–174.

(64)

Seib, F. P.; Pritchard, E. M.; Kaplan, D. L. Self-Assembling Doxorubicin Silk Hydrogels for the Focal Treatment of Primary Breast Cancer. Adv. Funct. Mater. 2013, 23, 58–65.

(65)

Shirosaki, Y.; Tsuru, K.; Hayakawa, S.; Osaka, A.; Lopes, M. A.; Santos, J. D.; Fernandes, M. H. In Vitro Cytocompatibility of MG63 Cells on Chitosan-Organosiloxane Hybrid Membranes. Biomaterials 2005, 26, 485–493.

(66)

Trujillo, S.; Pérez-Román, E.; Kyritsis, A.; Gómez Ribelles, J. L.; Pandis, C. OrganicInorganic Bonding in Chitosan-Silica Hybrid Networks: Physical Properties. J. Polym. Sci. Part B Polym. Phys. 2015, 53, 1391–1400.

(67)

Yang, J. M.; Shih, C. H.; Chang, C.-N.; Lin, F. H.; Jiang, J. M.; Hsu, Y. G.; Su, W. Y.; See, L. C. Preparation of Epoxy-SiO2 Hybrid Sol-Gel Material for Bone Cement. J. Biomed. Mater. Res. 2003, 64A, 138–146.

(68)

Li, G.; Chao, K.; Peng, H.; Chen, K. Hydrothermal Synthesis and Characterization of YVO4 and YVO4 :Eu3+ Nanobelts and Polyhedral Micron Crystals. J. Phys. Chem. C 2008, 112, 6228–6231.


Page 37 of 37

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TOC Figure


Fabrication of Biocompatible, Functional, and Transparent Hybrid

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